Serotonin [5-hydroxytryptamine, 5-HT] is a neurotransmitter, whose dysfunction is linked to the genesis of numerous psychiatric diseases, including mood disorders. The presynaptic serotonin transporters (SERTs) constitute the primary mechanism for re-uptake of 5-HT and termination of its signal in the synapse, and are high-affinity targets for addictive drugs including MDMA, cocaine and tricyclics, and SERT inhibitors constitute one of the largest groups of drugs used in the therapeutic treatment of depression. Little is known about the mode by which SERT function is regulated. Our preliminary data has identified a-synuclein, a presynaptic vesicle-associated protein of unknown function, as a modulator of SERT function, causing attenuation of normal transporter activity, indexed by diminished [3H]5HT uptake assays. The interaction between SERT and a-synuclein proceeds through the formation of stable protein:protein heteromeric complexes, and results in trafficking of SERT away from the cell surface of the plasma membrane. In this proposal we will study in detail the mechanisms which underline such regulation of transporter activity, using cells co-transfected with alpha-synuclein, and its subtypes, and the SERT cDNA, as well as in cultured primary neurons. In particular, the identity of the structural components which both facilitate and reverse a-synuclein effects on SERT function and trafficking, will be analyzed thoroughly, through a battery of studies to include, immunology, transporter assays, immunocytochemistry and FRET. We will also examine the participation of a-synuclein in both protein kinase C-mediated phosphorylation of SERT, and in transporter desensitization upon exposure to 5-HT. Our previous studies showing that a-synuclein can also modulate the function of the dopamine transporter, suggests that a primary function of this protein may be the regulation of monamine neurotransmitter homeostasis. Thus, our studies on SERT may have added heuristic and practical benefits for the understanding of other neurotransmitter transporters linked to mental illness and drug abuse. The identification of a-synuclein as a regulatory protein partner of SERT holds promise in the development of novel therapeutic strategies in the treatment of depression and drug addiction.